NIMA interacting proteins

ABSTRACT

A novel class of NIMA interacting proteins (PIN), exemplified by Pin1, is provided. Pin1 induces a G2 arrest and delays NIMA-induced mitosis when overexpressed, and triggers mitotic arrest and DNA fragmentation when depleted. Methods of identifying other Pin proteins and Pin-interacting proteins and identifying compositions which affect Pin activity or expression are also provided.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The present invention was made with government support from UnitedStates Public Health Services Grant No. CA 37980. The goverrnment hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the eukaryotic cell cycle andspecifically to a novel class of proteins that interact with NIMAprotein kinase in the NIMA mitotic pathway.

BACKGROUND OF THE INVENTION

The CDC2 kinase associated with its cyclin partners has been shown toplay an important role during G2/M progression in eukaryotic cells.However, recent studies demonstrate that activation of the CDC2 kinaseitself is not sufficient to trigger mitosis in some eukaryotic cellssuch as those in Saccharomyces cerevisiae (Amon, et al., Nature,355:368, 1992; Sorger and Murray, Nature, 355:365, 1992; Stueland etal., Mol. Cell. Bio., 13:3744, 1993) and Aspergillus nidulans (Osmani,et al., Cell, 67:283, 1991a). Furthermore, detailed analysis of mouseoocyte maturation reveals that CDC2 histone H1 kinase activity does notincrease during the G2/M transition as indicated by germinal vesiclebreakdown (GVBD) (Choi, et al., Development, 113:789, 1991; Jung, etal., Int. J. Dev. Biol., 37:595, 1993; Gavin, et al., J. Cell Sci.,107:275, 1994). These results suggest that there might be other mitoticactivation pathway(s) remaining to be identified.

Recent studies have identified a novel mitotic kinase, NIMA, encoded bythe Aspergillus nimA gene (Osmani, et al., Cell, 53:237, 1988). NlMAkinase activity is tightly regulated during the nuclear division cycle,peaking in late G2 and M. Overexpression of NIMA promotes entry ofAspergillus cells into M (Osmani, et al., Cell, 53:237, 1988; Lu andMeans, EMBO J., 13:2103, 1994). Thus, NIMA is important for progressioninto mitosis in Aspergillus.

NIMA is a protein-Ser/Thr kinase, biochemically distinct from otherprotein kinases, and its phosphotransferase activity is regulated bySer/Thr phosphorylation. It has recently been shown that the NIMAmitotic pathway is not restricted to Aspergillus, but also exists invertebrate cells (Lu and Hunter, Cell, 8:413, 1995a). In Xenopusoocytes, NIMA induces germinal vesicle breakdown without activating Mos,CDC2 or MAP kinase. In HeLa cells, NIMA induces mitotic events withoutactivating CDC2, whereas dominant-negative NIMA mutants cause a specificG2 arrest. In addition, O'Connell, et al (EMBO J. 13:4926, 1994) havealso demonstrated that NIMA induce premature chromatin condensation infission yeast and HeLa cells. These results reveal the existence of aNIMA-like mitotic pathway in other eukaryotic cells.

Peptidyl-prolyl cis/trans isomerases (PPIases, proline isomerases) areubiquitously expressed enzymes catalyzing the cis/trans isomerization ofthe peptidyl-prolyl peptide bond which can be the rate-limiting step inprotein folding or assembly under some circumstances. Cyclophilins andFK506-binding proteins (FKBPs) are two well characterized families ofPPIases that share little if any amino acid similarity to each other.However, the members of each family contain a core structure that hasbeen highly conserved from prokaryotes to eukaryotes. The importance ofthese PPIases is highlighted by the findings that cyclophilin andFK506-binding proteins are the targets of immunosuppressive drugscyclosporin A and FK506, respectively, and play an important role incell signaling in T cell activation, although none of these genes havebeen shown to be essential for life (for review see Schreiber, Science,251:283, 1991; Fruman, et al., FASEB J., 8:391, 1994).

The recent discovery of parvulin has led to the identification of athird family of PPIases, which show little homology with eithercyclophilins or FKBPs and is not sensitive to the immunosuppressivedrugs (Rahfeld, et al., FEBS Lett., 352:180, 1994a and FEBS Lett.,343:65, 1994b). A sequence homology search identified several othermembers of this family including those involved in protein maturationand/or transport and the ESS1 gene (Rudd, et al., TIBS, 20:12., 1995).ESS1 is an essential gene for the growth in budding yeast and previousresults suggested that it may be required at later stages of the cellcycle (Hanes, et al., Yeast, 5:55, 1989). ESS1 was recently reisolatedas PTF1 in a screen for genes involved in mRNA 3′ end maturation. Ptf1was shown to contain a putative PPIase domain, but PPIase activity couldnot be demonstrated (Hani, et al., FEBS Lett., 365:198, 1995). So far,none of the PPIases have been shown to be specifically involved in cellcycle control.

There is a need to identify components of the mammalian NIMA mitoticpathway in order to identify genes essential for life. Identification ofsuch genes has several utilities including the identification ofappropriate therapeutic targets, candidate genes for gene therapy (e.g.,gene replacement), mapping locations of disease-associated genes, andfor the identification of diagnostic and prognostic indicator genes, forexample.

SUMMARY OF THE INVENTION

The present invention provides a novel class of proteins that associatewith NIMA protein kinase. Some of these proteins are characterized byinhibiting the mitosis promoting function of NIMA when overexpressed andinducing mitotic arrest and nuclear fragmentation when depleted.

In a first embodiment, the invention provides an exemplary NIMAassociated protein called “protein interacting with NIMA” (Pin1). Pin1has C-terminal peptidyl-propyl cis/trans isomerase activity and containsa conserved N-terminal tryptophan domain (WW domain) thought to mediateprotein-protein interactions. Also included are polynucleotides encodingPIN proteins.

In another embodiment, the invention provides a method for identifying aprotein that inhibits the mitosis promoting function of NIMA proteinkinase. The method is based on a genetic system designed to detectprotein-protein interactions. The method comprises culturing transformedcells containing the following: a nucleic acid construct comprising aDNA binding domain operatively associated with the coding sequence ofNIMA, or functional fragments thereof; a nucleic acid library, whereineach member of said library comprises a transactivation domainoperatively associated with a protein encoding sequence; and a nucleicacid reporter construct comprising a response element for the DNAbinding domain operatively associated with a reporter gene, andmonitoring for evidence of expression of reporter gene.

In yet another embodiment, the invention provides a method forcontrolling the growth of a cell comprising contacting the cell with acomposition which modulates Pin1 activity. For examnple, an inhibitor ofPin1 activity such as a PPIase inhibitor or an anti-Pin1 antibody, or aninhibitor of PIN1 expression such as an antisense nucleotide sequence ora ribozyme, can be used to control growth of a cell. Alternatively, Pin1activity can be increased by an activator or PIN1 expression can beincreased by an enhancer, for example.

Finally, the invention provides a method for identifying a protein orother composition (e.g., drug or other small molecule) that associateswith and/or modulates Pin1 protein activity or PIN1 gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows human cDNA clones encoding proteins interacting with NIMA(Pins).

FIG. 1A shows a β-Galactosidase Activity in the two-hybrid system.

FIG. 1B shows a comparison of NIMA and NLK1.

FIG. 2 shows the cDNA and deduced amino acid sequences of human PIN1 andhomologies with other WW domain proteins and PPIases.

FIG. 2A shows the predicted Pin1 amino acid sequence is indicated inone-letter code. The fusion points between GAL4 and Pin1 in sixdifferent isolated clones were: clone H20 at C-9; clone H16, 24 and 38at G+13; clones H6 and H36 at C+15. Underlined residues form a consensusbipartite nuclear localization signal. The N- and C-terminal boxesindicate the WW domain and PPIase domain. Nucleotide numbers are on theleft and amino acid numbers on right.

FIG. 2B and 2C show alignments of the WW domain (B) and PPIase Domain(C) in selected proteins. Identical residues are shown in the bottomrow. Dashes indicate gaps introduced to make the alignment. Cbf2 cellbinding factor 2; SC, S. cerevisiae; EC, E. coli; BS, B. subtilis; CJ,C. jejuni; AT, A. thaliana.

FIG. 3 shows an analysis of PIN1 mRNA expression in human cell lines andhuman fetal liver. HeLa, epitheloid carcinoma cells; HFF, human foreskinfibroblasts; Ramos, Burkitt lymphoma cells; A431, epitheloid carcinomacells; 293; adenovirus E1A transformed human embryonic kidney cells;U937, histiocytic lymphoma cells; Saos-2, osteosarcoma cells; U87-MG,glioblastoma cells; Jurkat, T leukemia cell line and HFL, human fetalliver. Position of the RNA molecular weight standards (Promega) are onthe left.

FIG. 4 shows the PPIase activity of Pin1. Different concentrations ofPin1 were used as indicated, with the recombinant FK506-binding protein(FKBP) PPIase being used as a positive control. The control samplecontained all the ingredients except the PPIase. The insert shows PPIaseactivity during the first min of assay.

FIG. 5 shows immunoprecipitations to show the interaction between Pin1and the C-terminal noncatalytic domain of NIMAs in HeLa cells. FIG. 5Ashows the expression and purification of His-Pin1. The PIN1 cDNA wassubcloned into pProEx1 and the recombinant protein was purified frombacteria using NI²⁻-NTA agarose column, followed by analyzing theprotein on a 15% SDS-containing gel, and Coomassie staining. Thepositions of His-Pin1 and standard size markers are indicated. FIG. 5Bshows the expression of NIMAs in HeLa cells. tTA-1 cells weretransfected with different FLAG-tagged NIMA constructs and labeled with³⁵S Express Label for 24 hr, followed by immunoprecipitation using theM2 mAb. The precipitated proteins were analyzed on a 10% SDS-containinggel followed by autoradiography. The positions of K40M, NIMA, K40MNIMA280, NIMA280-699 and standard size mrarkers are indicated. FIG. 5Cshows a stable complex between recombinant Pin1 and NIMAs. Cell lysatessimilar to those in panel B were incubated with His-Pin1-Ni²⁻-NTA bead,as indicated in panel A and washed, followed by electrophoresis on anSDS-containing gel and autoradiography. FIGS. 5D and 5E showcoimmunoprecipitation of K40M NIMA with Pin1. tTA-1 cells werecotransfected with FLAG-tagged NIMA and HA-PIN1 constructs for 24 hr.Cell lysates were immunoprecipitated with the HA-specific 12CA5 mAb,followed by immunoblotting using the M2 mAB specific for the FLAG tag(D) and vice versa (E).

FIG. 6 shows the colocalization of Pin1 and NIMA and its kinase-negativemutant in HeLa Cells. Twenty four hr after transfection with the vectorsexpressing HA-tagged Pin1 only (right panels) or HA-tagged Pin1 andFLAG-taggged NIMA (left panels) or its kinase-negative mutant (K40MNIMA, middle panels), the transfected tTA-1 cells were processed forindirect immunofluorescence staining and examined by confocalmicroscopy. Top panels: staining pattern for NIMA or K40M NIMA obtainedwith FLAG tag-specific M2 mAb or SC-35 obtaining with anti-SC-35 mAb,and then FITC-conjugated IgG1-specific secondary antibodies; middlepanels: staining pattern for Pin1 obtaining with HA tag-specific 12CA5mAb and Texas-Red-conjugated IgG2b-specific secondary antibodies; bottompanels: double-staining for Pin1 and NIMA, Pin1 and K40M NIMA, or Pin1and SC-35 displayed by superimposing the respective top and middleimages, with yellow color indicating colocalization. Arrows point to anuntransfected cell, indicating very little cross reactivity among theantibodies. Bar, 1 μm.

FIG. 7 shows overexpression of PIN1 delays NIMA-induced mitotic arrestand induces a specific G2 arrest in HeLa cells. FIG. 7A shows theresults of tTA-1 cells cotransfected with nimA and PIN1 or controlvector. At the times indicated, cells were fixed and doubly labeled withM2 mAb and Hoechst dye, followed by scoring for the percentage of cellswith rounding and chromatin condensation in a sample of at least 250NIMA-expressing cells. The data are the average from two independentexperiments. FIGS. 7B, C, and D show after transfection with PIN1expression vector or the control vector for 48 hr, tTA-1 cells werestained with the 12CA5 mAb and then with FITC-conjugated secondaryantibodies and propidium iodide, followed by FACS analysis. Based on theFITC intensity of the PIN1-transfected cells, cells were divided intotwo populations with 12CA5-negative (B) or -positive cells (C), and cellcycle profiles were determined to compare with those in totalvector-transfected cells (A).

FIG. 8 shows depletion of Pin1/Ess1 results in mitotic arrest andnuclear fragmentation in yeast. A Pin1-dependent strain (YSH12.4) wasshifted from inducing media to repressing media, harvested and fixedwith 70% ethanol at the times indicated. The cells were stained withDAPI or propidium iodide, followed by videomicroscopy under Nomarksi(DIC) or fluorescent (DAPI) illumination, or FACS analysis,respectively. The bar is 10 μm and the inserts show a highermagnification of a representative cell.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A NIMA-like pathway is required for the G²/M transition in Aspergillusnidulans and human cells. The present invention provides the firstNIMA-interacting protein, Pin1, of mammalian origin, and methods foridentification of other NIMA-interacting proteins. Overexpression ofPIN1 and Pin1 activity, induces a specific G2 arrest and delaysNIMA-induced mitosis, while depletion of Pin1 triggers mitotic arrestand nuclear fragmentation in budding yeast.

In a first embodiment, the invention provides an isolated mammalianprotein characterized as associating with NIMA protein kinase,inhibiting the mitosis promoting function of NIMA when overexpressed,and inducing entry of cells into mitosis when depleted. The C-terminaldomain of such a protein catalyzes the cis/trans isomerization ofpeptidyl-propyl peptide bonds. The N-terminal domain associates withNIMA and contains at least two conserved tryptophan residues (WWdomain). While the exemplary polynucleotides and polypeptides of theinvention are directed to Pin1, it is understood that any PINpolynucleotide or Pin protein can now be identified and characterized bythe methods described herein.

In a preferred embodiment, the present invention provides asubstantially pure NIMA-interacting protein (Pin1) characterized byhaving a molecular weight of about 18kD as determined by reducingSDS-PAGE, having peptidyl-propyl cis/trans isomerase activity,associating with NIMA protein kinase, and having essentially the aminoacid sequence of SEQ ID NO:2. The term “substantially pure” as usedherein refers to Pin1 which is substantially free of other proteins,lipids, carbohydrates or other materials with which it is naturallyassociated. One skilled in the art can purify Pin1 using standardtechniques for protein purification. The substantially pure polypeptidewill yield a single major band on a non-reducing polyacrylamide gel. Thepuriry of the Pin1 polypeptide can also be determined by amino-terminalamino acid sequence analysis. Pin1 polypeptide includes functionalfragments of the polypeptide, as long as the activity of Pin1 remains.Smaller peptides containing one of the biological activies of Pin1 aretherefore included in the invention. Such peptides includeimmunologically reactive peptides capable of inducing antibodyproduction. The preferred Pin1 of the invention is derived from a humancell.

The invention provides isolated polynucleotides encoding Pinpolypeptides. including Pin1. These polynucleotides include DNA, cDNAand RNA sequences which encode Pin1. It is understood that allpolynucleotides encoding all or a portion of Pin1 are also includedherein, as long as they encode a polypeptide with Pin1 activity. Suchpolynucleotides include naturally occuring, synthetic, and intentionallymanipulated polynucleotides. For example, PIN1 polynucleotide may besubjected to site-directed mutagenesis. The polynucleotide sequence forPIN1 also includes antisense sequences. The polynucleotides of theinvention include sequences that are degenerate as a result of thegenetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in the invention as long as the amino acidsequence of Pin1 polypeptide encoded by the nucleotide sequence isfunctionally unchanged. Polynucleotides of the invention includevariations thereof which encode the same amino acid sequence, but employdifferent codons for some of the amino acids, or splice variantnucleotide sequences thereof.

Specifically disclosed herein is a DNA sequence encoding the human PIN1gene. The sequence contains an open reading frame encoding a polypeptide163 amino acids in length. The human PIN1 initiator methionine codonshown in FIG. 2A at position 25-27 is the first ATG codon. Preferably,the human PIN1 nucleotide sequence is SEQ ID NO:1 and the deduced aminoacid sequence is preferably SEQ ID NO:2 (FIG. 2A).

The polynucleotide encoding Pin1 includes SEQ ID NO:1 as well as nucleicacid sequences complementary to SEQ ID NO:1 (FIG. 2A). A complementarysequence may include an antisense nucleotide. When the sequence is RNA,the deoxynucleotides A, G, C, and T of SEQ ID NO:1 is replaced byribonucleotides A, G, C, and U, respectively. Also included in theinvention are fragments of the above-described nucleic acid sequencesthat are at least 15 bases in length, which is sufficient to permit thefragment to selectively hybridize to DNA that encodes the protein of SEQID NO:2 under physiological conditions. Specifically, the term“selectively hybridize” means that the fragments should hybridize to DNAencoding Pin1 protein under moderate to highly stringent conditions.

Minor modifications of the Pin1 primary amino acid sequence may resultin proteins which have substantially equivalent activity as compared tothe Pin1 polypeptide described herein. Such proteins include those asdefined by the term “having substantially the amino acid sequence of SEQID NO:2”. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein as long as the biologicalactivity of PIN still exists. Further, deletion of one or more aminoacids can also result in a modification of to the structure of theresultant molecule without significantly altering its biologicalactivity. This can lead to the development of a smaller active moleculewhich would have broader utility. For example, one can remove amino orcarboxy terminal amino acids which are not required for Pin1 biologicalactivity.

The Pin1 polypeptide of the invention encoded by the polynucleotide ofthe invention includes the disclosed sequence (SEQ ID NO:2; FIG. 2A) andconservative variations thereof. The term “conservative variation” asused herein denotes the replacement of an amino acid residue by another,biologically similar residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine, and thelike. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the PIN (e.g., PIN1) polynucleotide of the invention isderived from a mammalian organism, and most preferably from human.Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res.,. 9:879, 1981; Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989).

The development of specific DNA sequences encoding PIN can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; 3) in vitro synthesisof a double-stranded DNA sequence by reverse transcription of mRNAisolated from a eukaryotic donor cell; and PCR of genomic DNA or cDNAusing primers capable of annealing to the DNA sequence of interest. Inthe latter case, a double-stranded DNA complement of mRNA is eventuallyformed which is generally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepossible presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11 , can be screenedindirectly for PIN peptides having at least one epitope, usingantibodies specific for PIN. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of PIN1 cDNA.

DNA sequences encoding Pin1 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the PIN1 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of thePIN genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector tpically contains anorigin of replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg, et al.,Gene,56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryvelements, for example, a promoter (e.g., T7, metallothionein I,tetracycline responsive promoter or polyhedrin promoters).

Polynucleotide sequences encoding Pin1 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

WVhen the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the PIN of the invention, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.For example, one of skill in the art could use (His)₆ tag affinitypurification as described in the EXAMPLES herein.

The Pin polypeptides of the invention can also be used to produceantibodies which are immunoreactive or bind to epitopes of the Pinpolypeptides. Antibody which consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations are provided. Monoclonal antibodies aremade from antigen containing fragments of the protein by methods wellknown in the art (Kohler, et al., Nature, 256:495, 1975; CurrentProtocols in Molecular Biology, Ausubel, et al., ed., 1989).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)₂, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibocdies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York ( 1988), incorporated herein by reference).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to the Pin1 polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. For example, it may bedesirable to produce antibodies that specifically bind to the N- orC-terminal domains of Pin1. The polypeptide or a peptide used toimmunize an animal can be derived from translated cDNA or chemicalsynthesis which can be conjugated to a carrier protein, if desired. Suchcommonly used carriers which are chemically coupled to the peptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA), and tetanus toxoid. The coupled peptide is then used toimmunize the animal (e.g., a mouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

In another embodiment, the invention provides a method for identifying aprotein that inhibits the mitosis promoting function of NIMA proteinkinase. The method comprises culturing transformed cells containing thefollowing: a nucleic acid construct comprising a DNA binding domainoperably associated with the coding sequence of NIMA, or functionalfragments thereof; a nucleic acid library, wherein each member of saidlibrary comprises a transactivation domain operably associated with aprotein encoding sequence; and a nucleic acid reporter constructcomprising a response element for the DNA binding domain operablyassociated with a reporter gene, and monitoring for evidence ofexpression of reporter gene.

The term “inhibits” refers to a reduction in the mitosis promotingfunction of NIMA protein kinase. The “mitosis promoting function ofNIMA” refers to the ability of NIMA to promote the progression of theG2/M transition in a cell. The term “operably associated” refers tofunctional linkage between the promoter or regulatory sequence and thecontrolled nucleic acid sequence; the controlled sequence and regulatorysequence, or promoter are typically covalently joined, preferably byconventional phosphodiester bonds.

The method of the invention comprises culturing cells, under suitableconditions, transformed by standard methods in the art and as describedabove. The transformed cells contain the followint: a nucleic acidconstruct comprising a DNA binding domain operably associated with thecoding sequence of NIMA, or functional fragments thereof. As usedherein, the term “DNA binding domain” refers to a nucleic acid sequencewhich contains a recognition site for a protein that binds to specificDNA sequences. The coding sequence of NIMA includes nucleic acidsequence that encodes a protein having the biological activity of NIMAas described herein. The method includes “functional fragments” of NIMAthat are less than the full length coding sequence but which encode aprotein having NIMA biological activity (e.g., Ser/Thr protein kinase).

The transformed cells also contain a nucleic acid library, wherein eachmember of the library comprises a transactivation domain operablyassociated with a protein encoding sequence. A “transactivation domain”refers to a nucleic acid sequence which activates transcription, buttypically fails to bind to DNA. A nucleic acid library consists of aclone bank of genomic or complementary DNA sequences which are “proteinencoding sequences”. The term refers to cDNA operably associated withthe transactivation domain of a protein as exemplified in the EXAMPLES.

The cells also contain a nucleic acid reporter construct comprising aresponse element for the DNA binding domain operably associated with areporter gene. As used herein, the term “reporter” refers to a geneencoding a trait or a phenotype which permits the monitoring andselection of, or the screening for, a cell containing the marker, andthus a NIMA interacting protein. The nucleic acid reporter is aselectable marker such as a color indicator (e.g., lacZ). Other suitablereporter genes will be known to those of skill in the art.

The protein-protein interaction system exemplified in the EXAMPLESherein preferably utilize the GAL4 protein to provide the DNA bindingand transactivation domains (Fields and Song, Nature, 340:245, 1989).Other nucleic acid constructs comprising the coding sequences ofproteins that have a DNA binding and transactivation domain will beknown to those of skill in the art and can be utilized in the method ofthe invention for providing the hybrid constructs.

The method of the invention relies on interaction between the libraryencoded protein and the NIMA protein, or functional fragment thereof, inorder to activate transcription of the reporter gene. Once the libraryencoded protein and the NIMA protein associate, they bring the DNAbinding domain and the transactivation domain in close proximity,resulting in transcriptional activity. Thus, the method provides a meansfor identifying a protein that interacts or associates with NIMA proteinkinase.

While the method of the invention as described above is preferred, it isunderstood that the transformed cell may contain the reverse constructs,e.g., a nucleic acid construct comprising a transactivation domainoperably associated with the coding sequence of NIMA, or fragmentsthereof, and a nucleic acid library, wherein each member of said librarycomprises a DNA binding domain operably associated with a proteinencoding sequence.

Also included in the present invention are NIMA interacting proteinsidentified by the above method.

The invention also provides a method for controlling the growth of acell comprising contracting the cell with a composition which modulatesPin1 activity. The term “modulate” envisions the suppression ofexpression of PIN1 or suppression of Pin1 activity, when it isover-expressed, or augmentation of PIN1 expression or Pin1 activity whenit is under-expressed. Growth of a cell is “controlled”, for example, byinhibiting mitosis promoting function of NIMA or inducing entry of cellsinto mitosis. An inhibitor of Pin1 activity or protein level, forexample, would result in arrest in mitosis. Therefore, Pin1 activity orprotein level can be decreased, leading the cell to mitotic arrest, oralternatively, Pin1 activity or protein level can be increased,arresting the cells in G2.

Where controlling the growth of a cell is associated with the expressionof PIN1 polynucleotide, nucleic acid sequences that interfere with PIN1expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid, ribozymes, or triplexagents to block transcription or translation of a specific PIN mRNA,either by masking that mRNA with an antisense nucleic acid or triplexagent, or by cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids primarily work via RNaseH-mediateddegradation of the target mRNA. Antisense may also interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetPIN-producing cell. The use of antisense methods is well known in theart (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991: Helene. C., Anticancer Drug Design,6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

Other Pin inhibitors include PPIase inhibitors. For example,immunosuppressive drugs such as cyclosporin A and FK506, are PPIaseinhibitors useful in the method of the invention for inhibiting some Pinproteins. The Pin1 or other Pin protein of the invention is useful forscreening for other inhibitors as well. For example, a suspected PPIaseinhibitor is incubated with Pin1 protein under suitable conditions toallow Pin1 enzymatic activity to be expressed and measured, and thelevel of activity in the presence and absence of the inhibitor isassayed to determine the effect on Pin1 activity.

Pin1 activity may also be increased in the presence of an activator.PIN1 expression may be increased in the presence of an enhancer forexample. Stimulation of Pin1 activity or overexpression of PIN1 arreststhe cells in G2 and inhibits the mitosis promoting function of NIMA. Thecis-acting elements which control genes are called promoters, enhancersor silencers. Promoters are positioned next to the start site oftranscription and function in an orientation-dependent manner, whileenhancer and silencer elements, which modulate the activity ofpromoters, are flexible with respect to their orientation and distanceto the start site of transcription. One of skill in the art will knowmethods for stimulating expression of PIN1 by including an enhancer, forexample, to stimulate PIN1 expression. The effect of an enhancer orother regulatory element can be determined by standard methods in theart (e.g., Northern blot analysis, nuclear runoff assay).

Pin1 activity can be affected by an activator. An “activator” as usedherein includes a protein or a small molecule, such as an organiccompound, for example, which increases Pin1 activity or protein level,such that a cell arrests in G2. An activator can be identified byincubating Pin1 and a putative activator under conditions that allowinteraction between the components, and measuring of the effect of theactivator on Pin1. For example, one could determine whether cells werearrested in G2 (e.g., increased Pin1 activity), or whether cellscontinued to cycle through G2 to M (e.g., by FACS analysis or labelednuclei analysis).

The Pin1 protein of the invention is useful in a screening method toidentify compounds or compositions which affect the activity of theprotein or expression of the gene. Thus, in another embodiment, theinvention provides a method for identifying a composition which affectsPin1 comprising incubating the components, which include the compositionto be tested (e.g., a drug or a protein) and Pin1, under conditionssufficient to allow the components to interact, then subsequentlymeasuring the effect the composition has on Pin1 activity or expression.The observed effect on Pin1 may be either inhibitory or stimulatory. Forexample, the entry of cells into mitosis or G2 arrest can be determinedby nuclear content (e.g., immunofluorescence or FACS analysis based onDNA content) or other methods known to those of skill in the art.

The increase or decrease of PIN1 transcription/translation can bemeasured by adding a radioactive compound to the mixture of components,such as ³²P-ATP (for nuclei) or ³H-Uridine, or ³⁵S-Met, and observingradioactive incorporation into PIN1 transcripts or protein,respectively. Alternatively, other labels may be used to determine theeffect of a composition on PIN1 transcription/translation. For example,a radioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme could be used.Those of ordinary skill in the art will know of other suitable labels orwill be able to ascertain such, using routine experimentation. Analysisof the effect of a compound on PIN1 is performed by standard methods inthe art, such as Northern blot analysis (to measure gene expression) orSDS-PAGE (to measure protein product), for example. Further, Pin1biological activity can also be determined, for example, byincorporation label into nuclei.

The method of the invention described above also includes identifying aprotein that associates with Pin1 protein. The method comprisesincubating the protein with Pin1 protein, or with a recombinant cellexpressing Pin1, under conditions sufficient to allow the components tointeract and determnining the effect of the protein on Pin1 activity orexpression. Alternatively, one of skill in the art could use thetwo-hybrid system described above to identify a protein that interactsor associates with Pin1 protein. Once identified, suchPin/PIN-associated proteins may now be used as targets for drugdevelopment.

In another embodiment, the invention provides a method for treating acell proliferative disorder. The method comprises administering asubject in need of such treatment, an amount of Pin1 inhibitor effectiveto induce entry of cells into mitosis or apoptosis. The “amount of Pin1inhibitor effective to induce entry of cells into mitosis” means thatthe amount of polypeptide, peptide, polynucleotide, or monoclonalantibody for example, which when used, is of sufficient quantity toameliorate the disorder. The term “cell-proliferative disorder” denotesmalignant as well as non-malignant cell populations whichmorphologically often appear to differ from the surrounding tissue bothmorphologically and genotypically. Malignant cells (i.e. cancer) developas a result of a multistep process. The PIN1 polynucleotide that is anantisense molecule is useful in treating malignancies of the variousorgan systems. For example, the method may be useful in treatingmalignancies of the various organ systems, such as, for example, lung,breast, lymphoid, gastrointestinal, and genito-urinary tract as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer, leukemia, breast cancer,non-small cell carcinoma of the lung, cancer of the small intestine, andcancer of the esophagus. Essentially, any disorder which isetiologically linked to altered expression of PIN1 could also beconsidered susceptible to treatment with a PIN1 suppressing/inhibitingreagent.

The method is also useful in treating non-malignant orimmunologically-related cell-proliferative diseases such as psoriasis,pemphigus vulgaris, Bechet's syndrome, acute respiratory distresssyndrome (ARDS), ischemic heart disease, post-dialysis syndrome,rheumatoid arthritis, acquired immune deficiency syndrome, vasculitis,lipid histiocytosis, septic shock and inflammation in general.Essentially, any disorder which is etiologically linked to PIN1 wouldalso be considered susceptible to treatment.

For purposes of the invention, an antibody or nucleic acid probespecific for Pin1 may be used to detect Pin1 polypeptide (usingantibody) or polynucleotide (using nucleic acid probe) in biologicalfluids or tissues. The invention provides a method for detecting a cellproliferative disorder which comprises contacting an anti-Pin1 antibodyor nucleic acid probe with a cell suspected of having a Pin1 associateddisorder and detecting binding to the antibody or nucleic acid probe.The antibody reactive with Pin1 or the nucleic acid probe is preferablylabeled with a compound which allows detection of binding to Pin1. Anyspecimen containing a detectable amount of antigen or polynucleotide canbe used. The level of Pin1 in the suspect cell can be compared with thelevel in a normal cell to determine whether the subject has aPin1-associated cell proliferative disorder. Preferably the subject ishuman.

When the cell component is nucleic acid, it may be necessary to amplifythe nucleic acid prior to binding with an PIN1 specific probe.Preferably, polymerase chain reaction (PCR) is used, however, othernucleic acid amplification procedures such as ligase chain reaction(LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes viris, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). Preferably, when the subject is a human, a vectorsuch as the gibbon ape leukemia virus (GaLV) is utilized. A number ofadditional retroviral vectors can incorporate multiple genes. Otherviral vectors include DNA vectors such as adenovirus andadeno-associated virus (AAV). All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a PIN1 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Preferredtargeting is accomplished by usin an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome or attached to a viralenvelope to allow target specific delivery of the retroviral vectorcontaining the PIN1 antisense polynucleotide. In addition, the viralvector may contain a regulatory element such as a tetracyclineresponsive promoter (inducible or repressible) operably linked to apolynucleotide sequence).

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packazing signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Another targeted delivery system for PIN1 polynucleotides (e.g.,antisense) is a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preferred colloidal systemof this invention is a liposome. Liposomes are artificial membranevesicles which are useful as delivers vehicles in vitro and in vivo. Ithas been shown that larvae uni-lamellar vesicles (LUV), which range insize from 0.2-4.0 μm can encapsulate a substantial percentage of anaqueous buffer containing large macromolecules. RNA, DNA and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form (Fraley, et al., Trends Biochem.Sci., 6:77, 1981). In addition to mammalian cells, liposomes have beenused for delivery of polynucleotides in plant, yeast and bacterialcells. In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficienct while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Materials and Methods

1. Yeast Two-Hybrid Screen and cDNA Isolation

For the yeast two-hybrid screen, the Aspergillits nidulans nimA cDNA wasinserted into the pAS2 vector (from S. Elledge; HHMI, Baylor University)(Durfee et al., 1993; Harper, et al., 1993) as fusion to GAL4DNA-binding domain, resulting in NIMA/PAS2. A HeLa cell two-hybrid cDNAlibrary that contained inserts fused to the transactivation domain ofGAL4 (residue 768-881) (obtained from D. Beach; HHMI, Cold Spring HarborLaboratory, New York Nov. 12, 1995) (Hannon et al., 1993) wascontransformed with NIMA/PAS2 into the yeast reporter strain Y190, whichwas then plated on yeast drop-out media lacking Leu. Trp and Hiscontaining 35 mM 3-amino-1,2,4 triazole and about 10⁶ transformants wereanalyzed as described previously (Harper et al., 1993). To determine theinteraction properties of isolated clones, K40M NIMA, NIMA280-699, NLK1and a frame-shift mutant NIMA280-699^(FS) (a base pair deletion at thelitigation point) were also subcloned into the pAS2 vector, followed bycotransformation into Y190 cells with the isolated cDNA clones. Toobtain additional PIN1 5′ untranslated sequence, a HeLa cell cDNAlibrary constructed by R. Fukunaga; Salk Institute, La Jolla, Calif.)was screened with the H20 cDNA fragment. DNA sequence was determined bythe dideoxynucleotide chain termination method.

2. Isolation of Human NIMA-Like Kinase 1 (NLK1)

For polymerase chain reaction (PCR), three degenerate oligonucleotidesGCGCCTGCAGTATCTATAC/TATGGAATAT/CTGT/(SEQ ID NO: 3)GCGCGGATCCG/AGGTTTCAGAGGT/GTC/TG/AAAG/CAG (SEQ ID NO: 4) andGCGCGTACCAAGT/ACCACT/CGTAC/TATTATTCC (SEQ ID NO: 5) were designedspecifically to the catalytic domain V, VII and VIII, respectively,which are conserved among NIMA (Osmani et al., Cell. 53:237, 1988), NPK,a budding yeast NIMA-related kinase (Schweitzer and Philippsen, Mol.Gen. Geneti., 34:164, 1992) and human HsPK2 (Schultz and Nigg, J. Cell.Growth Differ., 4:821, 1993). Human placenta cDNA library (from R.Evans, Salk Institute, La Jolla, Calif.) was used as a template. The PCRcycle was 1 min at 95° C., 2 min at 42° C. and 3 min at 63° C. PCRproducts with the expected size were subcloned and sequenced. Threepotential clones (Ping-1, -44 and -77) were consistently obtained andshowed a high sequence identity to NIMA at the deduced amino acids. Toobtain the full length cDNA sequence, the Ping-1 PCR product was used asa probe to screen HeLa cell cDNA library filters provided by X. D. Fu(Gui et al., Nature, 369:678, 1994); over thirty positive clones wereobtained and encode the same protein, referred to as NLK1 (NIMA-likekinase 1). NLK1 cDNA is 21521 bp and encodes 445 amino acids with anapparent molecular weight of 45 kDa on a SDS-polyacrylamide gel. NLK1was found to be same as NEK2 reported later by Schultz et al. (Cell.Growth Differ., 5:625, 1994)

3. DNA Transfection and Indirect Immunofluorescence Microscopy

NIMA and its derivative mutant expression constructs were the same asdescribed previously (Lu and Hunter, Cell. 81:413, 1995a). Forexpression of Pin1, an HA tag (MYDVPDYASRPQN) (SEQ ID NO:6) was added tothe N-terminus of PIN1 clones H20 and H6, followed by insertion intopUHD 10-3 vector as described previously (Lu and Means, EMBO J.,13:2103, 1994). Transfection of the tTA-1 cell line (Gossen and Bujard,Proc. Natl. Acad. Sci. USA, 89:5547, 1992) was described previously(Elredge et al., Meth. Enzymol., 254:481, 1995) with the exception thatcells were plated at lower density (30%), which seemed to increase thetransfection efficiency and the percentage of cycling cells. Indirectimmunofluorescence microscopy was performed as described (Lu and Hunter,Cell, 81:413, 1995a). The dilution of primary antibodies was: mouse M2mAb (Kodak/IBI.IgaG1 isotype), 1:600; 12CA5 mAb (IgG2b isotype, 1:1500:SC35 mAb (from X. Fu. IgG1 isbtype) (Fu and Maniatis, Nature, 343:437,1990); straight supernatant; and rabbit anti-PML antibodies (from R.Evans) (Dyck et al., Cell. 76:333, 1994), 1:100. The FITC-conjugatedgoat anti-mouse IgG1 and Texas Red-conjugated goat anti-mouse IgG2bsecondary antibodies (Southern Biotechnology) were used at 1:50, with nosignificant cross-reactivity. Cells were observed and photographed witha Zeiss microscope, or an MRC-1000 laser-scanning confocal assembly(BioRad).

4. Flow Cytometry Analysis

Cells were harvested 48 hr after transfection and stained with the 12CA5MAb, followed by flow cytometry analysis on a Becton-Dickinson FACScanmachine (Lu and Hunter, Cell, 81:413, 1995a). 12CA5-negative or-positive cells (10⁴) were collected to determine the DNA content, andcell cycle profiles were determined using the M Cycle analysis software(Phoenix Flow Systems, San Diego, Calif.).

5 . Metabolic Labeling, Immunoprecipitation and Immunoblot Analysis

Metabolic labeling of tTA-1 cells was as described previously (Lu andHunter, Cell. 81:413, 1995a). Cells were lysed in a NP40 lysis buffer(50 mM Tris-HCl, pH 8.0, 0.1% NP40, 200 mM NaCl, 20 mMβ-glycerophosphate, 20 mM NaF, 0.1 mM sodium orthovanadate, 50 μg/mlphenylmethylsufonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin,and 1 mM DTT or 10 mM β-mercaptoethanol (in the case of Ni²⁻-NTA agaroseprecipitation) and preclarified with boiled S. aureis or Ni²⁻-NTAagarose (Qiagen). The lysates were then incubated with M2or 12CA5 orHis-Pin1-Ni²)⁻-NTA agarose. After washing 6 times with lysis buffer, theprecipitates were subjected to SDS-polyacrylamide, gel electrophoresisand immunoblotting.

6. Expression and Purification of Pin1 and Kinase Assays

To express the (His)₆ tag-containing Pin1 protein, the cDNA insert fromH20 clone was subcloned into SpeI/HindIII sites of pProEx1 I and therecombinant protein was expressed in and purified from BL21 bacterialstrain using Ni²⁻-NTA agarose column (Qiagen) as described by themanufacturer. To examine whether Pin1 could be phosphorylated,recombinant Pin1 protein was added to NIMA kinase reaction mixtures atconcentrations up to 0.5 mg/ml, with β-casein and PL1 pepcide aspositive controls, as described previously (Lu et al., J. Biol. Chem.,269:6603, 1994). The effect of Pin1 on NIMA was assayed using PL1 as asubstrate with increasing concentrations of recombinant Pin1 (Lu et al.,J. Biol. Chem., 269:6603, 1994). Cyclin B/CDC2 (obtained from N.Watanabe; Salk Institute) ERK1 MAPK (obtained from R. Fukunaga) and PKA(Promega) were assayed using histone H1, myelin basic protein and H1 assubstrates respectively.

7. PPIase Assay

PPIase activity of the purified recombinant proteins was assayed usingthe procedure of Heitman et al. (Methods, 5:176, 1993) with thefollowing exceptions. The reaction was carried out 5° C. andchymotrypsin was added just betore the peptide substrate(N-Succ-Ala-Pro-Phe-p-nitroanilide, Sigma). The cis to transisomerization was monitored every 6 seconds by a change in theabsorbance at 395 nm using DMS 2000 UV and Visible Spectrophotometer(Varian). FKBP, cyclosporin A and FK520, a FK506 derivative, were giftsof D. Schultz and C. Zuker, University of California, San Diego.

8. Yeast Complementation Assays

PIN1 expression was driven from the strong constitutive yeast triosephosphate isomerase promoter (TPI) (Smith et al., 1985). PlasmidpTPI-PIN1 was made by insertion of an ˜850 bp BamHI-XhoI fragment ofPIN1 cDNA (from H20/GADGH) into pJK305-TPI (J. Kamens, Ph.D. Thesis,Harvard University, 1991). PTPI-PIN1 directs the synthesis of the nativefull length Pin1 protein and carries the yeast 2μ replicator and LEU2selectable marker. Plasmid YepHESS carries the yeast ESS1 gene, a 2μreplicator and HIS3 selectable marker (Hanes et al., Yeast, 5:55, 1989).

For tetrad analysis, a heterozygous ESS1 disruption strain MGG3/pSH-U(MATa/MATα ura3/ura3 leu2/ leu2 his3/ his3 ess1::URA3/ESS1) (Hanes etal., Yeast, 5:55, 1989), was transformed with control vector pJK305-TPI,YepHESS, or pTPI-PIN1. Cells were induced to undergo sporulation on 1%potassium acetate plates, tetrads were dissected and haploid segregantsgrown for 3-4 days at 30° C. on rich medium (YEPD) plates. Only tetradsthat showed proper segregation of the URA3, and MATa and MATα alleleswere included in the viable spore counts. For the curing experiment, ahomozygous disruption derivative of MGG3/pSH-U (relevant genotypeess1::URA3/ess1::URA3) carrying ESS1 on episomal plasmid (YepHESS) wastransformed with either pJK305-TPI or pTP1-PIN1. Cells were seriallypassaged once per day (1/50 Dilutions) for 6 days in liquid completersynthetic medium lacking leucine; thus maintaining selection for thePIN1-expressing plasmid or control vector (2μ, LEU2), but not for theESS1-bearing plasmid (2 μM, HIS3). Cells were plated and phenotypes ofindividual colonies scored by replica plating to appropriate selectivemedia.

9. PIN1-Decendent Yeast and Determination of Terminal Phenotype

For Pin1 depletions experiments in yeast, we used the GAL1 promoter(Yocum et al., Mol. Cell. Bio., 4:1985, 1984). Plasmid pGAL-PIN1, wasmade by insertion of a SpeIXohI fragment of the PIN1 cDNA frompN2P1/GADGH into AvrII and XhoI cut pBC103. PGAL-PIN1 directs thesynthesis of an HA1 hemagglutinin epitope-tagged version of Pin1 thatcontains a 31 amino-terminal extension MASYPYDVPDYASPEFLVDPPGSKNSIAGMK)(SEQ ID NO:7) where underlined residues are the HA1 epitope and thenormal PIN1 initiator, respectively. Other residues derive frompolylinker sequences. An integrating version, I-GAL-PIN1, was made byremoval of the GAL-PIN1 cassette from pGAL-PIN1 with Xba1 and SacI andreinsertion into the same sites in pRS305 (Sikorski and Hieter,Genetics. 122:19, 1989). I-GAL-PIN1 carries a LEU2 selectable marker.

The original ess1 knockout strain (MGG3/pSH-U) does not grow ingalactose, probably due to a gal2 mutation. Therefore, to make a strain(YSH12) that lacks ESS1 function and whose growth depends on agalactose-inducible version of PIN1, the following scheme was used. Agal strain, FY86 (MATa his3

200 ura3-52 leu2

1) was transformed with integrating vector GAL-PIN1 (LEU2). Leutransformants were mated with a haploid MGG3/pSH-U segregant (MATa ura3his3 leu2 ess1::URA3) carrying YEpHESS. Diploids were sporulated andtetrads dissected on rich medium containin 2% galactose/1% raffinose.Haploid segregants that contained the ess1::URA3 disruption (ura) andGAL-PIN1 (leu), but lacked YEpHESS (his) were identified. Isolates thatshowed growth on galactose (YEPG) but not on glucose (YEPD) medium erefurther characterized. Cells that did not contain the ess1 knockout(ura) but did contain GAL-PIN1 (leu) were used as controls. Inducibleexpression of PIN1 in several isolates of YSH12 was confirmed byimmunoblot analysis.

To shut off expression of PIN1 in ess1 cells, strain YSH12 was grownovernight in galactose or galactose/glucose-containing medium andinoculated (˜1/50 dilution) into glucose containing-medium. Cells weregrown for approximately 12 hours and then reinoculated into freshglucose-containing medium for an additional 18 hours. Aliquots of cellswere harvested by centrifugation, fixed by the addition of 70% ethanoland stored at 4° C. Bud size distribution was scored under DIC(Normarski) illumination after cells were resuspended in water andsonicated extensively to disperse clumped cells. For DAPI staining, thefixed cells were stained with 1 μg/ml DAPI and mounted in 75% glyceroland photographed under fluorescent illumination. FACS analysis of yeastcells was described previously (Sazer and Sherwood, J. Cell Sci., 509,1990).

EXAMPLE 2 Identification of Clones Encoding Proteins Interacting WithNIMA (PINS)

To search for human cDNAs encoding proteins able to interact with NIMA,a yeast two-hybrid system that uses GAL4 recognition sites to regulateexpression of both HIS3 and LacZ (Durfee et al., Genes Dev., 7:555,1993) was used. As bait, the full length coding sequence of theAspergillus nidulans nimA was fused to the C-terminus of the GAL4DNA-binding domain in pAS2 driven by a partially ADH promoter (Harper,et al., Cell, 75:805, 1993). As prey, we used the GAL4 transactivationdomain and a human HeLa cell cDNA fusion library driven by the highlyactive entire ADH promoter, resulting in high level expression (Hannon,et al., Genes Dev., 2378, 1993). Initially, Y190 cells were transformedwith the NIMA/pAS2 expression vector in an attempt to establish stablestrains constitutively expressing NIMA, but the transformant colonieswere very small and could not be propagated. The failure of thetransformants to grow is probably due to the fact that NIMA inducesmitotic arrest in budding yeast, as it does in the other differenteukaryotic cells so far examined (Osami, et al., Cell, 53:237, 1988: Luand Means, EMBO J., 13:2103, 1994; O'Connell et al., EMBO J., 13:4926,1994: Lu and Hunter, Cell, 81:413, 1995a). To circumvent this problem,Y190 yeast cells were cotransformed with NIMA/pAS2 and the two-hybridscreen cDNA library in the hope that expression of one or more of thecDNA library products might rescue the lethal phenotype of NIMA.

Out of 10⁶ colonies screened, 13 were consistently positive upon repeattransformations. The specificity of the interaction was tested usingfull length NIMA, a C-terminal noncatalytic fragment of NIMA,NIMA280-699, and a human NIMA-like kinase 1 (NLK1) (FIG. 1A). FIG. 1Ashows a β-Galactosidase activity in the two-hybrid system. Yeast strainY190 was cotransforned with vectors expressing the three different typesof cDNA and different domains of NIMA or NLK1 as indicated, and grown inSC media lacking Trp and Leu, followed by β-galactosidase activityfilter assay as described previously (Durfee, et al., Genes Dev., 7:55,1993). NLK1, which was isolated from a human placenta cDNA library andproved to be the same as Nek2 (Schultz et al., Cell Growth Differ.,5:625, 1994), is 47% identical to NIMA in its catalytic domain, but NIMAand NLK1 has very little similarity in their C-terminal noncatalyticdomains (FIG. 1B). Based on the sequences of their inserts and thespecificities of their interaction with NIMA and NLK1, these clones fellinto three different cene classes, referred to as PIN1, PIN2 and PIN3(PIN=protein interacting with NIMA). There were six PIN1, three PIN2 andfour PIN3 clones. The analysis of the two-hybrid interactions indicatePin3 interacts with the catalytic domains in both NIMA and NLK1s,whereas Pin1 and Pin2 interact with the C-terminal domain of NIMA. Thefollowings examples focus on Pin1 as an exemplary Pin protein.

EXAMPLE 3 Analysis of the PIN1 cDNA

The DNA and deduced amino acid sequence of the protein encoded by thelongest PIN1 cDNA (1.0kb) is shown in FIG. 2A. It encodes a protein of163 amino acids with the predicted molecular weight of 18,245. Whenexpressed in HeLa cells, Pin1 migrated with an apparent size of ˜18 kDain an SDS-polyacrylamide gel. Pin1 shows a high similarity to thebudding yeast Ess1 protein (Hanes et al., Yeast, 5:55, 1989). Based onrecent RNA primer-extension studies and reexamination of the nucleotidesequence of ESS1 (Hani et al., FEBS Lett., 365:198, 1995), it is likelythat the second ATG is used as the initiation codon, rather than thefirst ATG as originally proposed (Hanes et al., Yeast, 5:55, 1989); inaddition, a G should be inserted at position 919, which results in aC-terminal frame shift. The corrected ESS1 sequence encodes a 169 aminoacid protein with 45% identity to Pin1. Northern blot analysis of humancell line and human fetal liver RNAs showed that there is a single PIN1mRNA of ˜1.0kb present in all cell lines and tissue tested (FIG. 3).Fifteen μg of the indicated total RNAs wsere run on each lane, HeLa,epitheloid carcinoma cells: HFF, human foreskin fibroblasts: Ramos,Burkitt lymphoma cells; A431, epitheloid carcinoma cells; 293;adenovirus E1A transformed human embryonic kidney cells; U937,histiocytic lymphoma cells; Saos-2, osteosarcoma cells; U87-MG,glioblastoma cells; Jurkat, T leukemia cell line and HFL, human fetalliver. Position of the RNA molecular weight standards (Promega) are onthe left.

These results, together with the sequence comparison between Pin1 andEss1, confirm the authenticity of the PIN1 open reading frame, althoughthere is no inframe termination condon preceding the putative initiationsite. Sequence analysis of six different PIN1 cDNA clones identified thepoints of fusion with the GAL4 activation domain at Arg=(−3), Glu+(+5)and Lys−(+6), indicating that the N-terminal 5 amino acids of Pin1 arenot necessary for the interaction with NIMA. Immunoblot analysis usinganti-Pin1 antibodies confirmed that Pin1 is an 18 kD protein in thecell.

The deduced Pin1 sequence contains two identifiable domains, anN-terminal WW domain and C-terminal putative PPIase domain. The WWdomain contains two invariant Trp residues and other residues highlyconserved in the WW domains of other proteins including mammaliandystrophins and Yap, and the yeast Rsp1 and Ess1 (Sudol et al., 1995).However, the Cys or His-rich domain flanking the WW domain found in WWdomain-containing proteins including Ess1 (Sudol et al., 1995) is notconserved in the human Pin1. The C-terminal two-thirds of Pin1 containsmotifs that are characteristic of the recently identified third familyof PPIases (Rudd et al., TIBS, 20:11, 1995). The PPIase domain of Pin1contains three highly observed subdomains, with 45% identity to parvulinand 62% identity to a partial PPIase from A. thaliana. A putativenuclear localization signal is located in the beinning of the PPIasedomain, which is also conserved in Ess1.

FIG. 2A shows the predicted Pin1 amino acid sequence is indicated inone-letter code. The fusion points between GAL4 and Pin1 in sixdifferent isolated clones were: clone H20 at C-9; clone H16, 24 and 38at G+13; clones H6 and H36 at C-31 15. Underlined residues form aconsensus bipartite nuclear localization signal. The N- and C-terminalboxes indicate the WW domain and PPIase domain. Nucleotide numbers areon the left and amino acid numbers on right. FIGS. 2B and 2C showalignments of the WW domain (B) and PPIase Domain (C) in selectedproteins. Identical residues are shown in the bottom row. Dashesindicate gaps introduced to make the alignment. Cbf2, cell bindinrgfactor 2; SC, S. cerevisiae; EC, E. coli; BS, B. subtilis; CJ, C.jejuni; AT, A. thaliana.

EXAMPLE 4 Pin1 HAS PPIase Activity

Because of the strong sequence similarity between Pin1 and the thirdPPIase family, Pin was tested to determine if Pin could catalyze thecis/trans isomerization of peptidyl-prolyl peptide bonds in vitro, acharacteristic of PPIase. Pin1 was expressed in bacteria with anN-terminal (His)₆ tag and was purified from bacteria usinl, a Ni²⁻-NTAagarose column. When purified recombinant Pin1 was tested for PPIaseactivity using a standard chymotrypsin-coupled spectrophotometric assay(Heitman et al., Methods, 5:176, 1993), PPIase activity was readilydetected which was highly concentration-dependent, with a specificactivity similar to that of recombinant FKBP (FIG. 4). The isomeraseactivity was measured as described in Example 1. The cis to transisomerization was monitored every 6 seconds by a change in theabsorbance at 395 nm, with the finally stable absorbance of each samplebeing set at 1.0. Different concentrations of Pin1 were used asindicated, with the recombinant FK506-binding protein (FKBP) PPIasebeing used as a positive control. The control sample contained all theingredients except the PPIase. The insert shows PPIase activity duringthe first minute of the assay (▴control; ◯Pin1, 71 μg/ml; □Pin1, 14μg/ml; ▪Pin1, 7 μg/ml; ●FKBP 70 μg/ml).

Like parvulin, the PPIase activity of Pin1 was not inhibited by eithercyclosporin A or FK520, a derivative of FK506, even at 25 μM. Theseresults contirm that Pin1 is a to member of the third family of PPIase.

EXAMPLE 5 Pin1 Interacts With the C-Terminal Domain of NIMA

In the two-hybrid system, Pin1 interacted with NIMA, kinase-negativeK40M NIMA and C-terrninal NIMA280-699, but not with NLK1 (Table 1),indicating that NIMA interacts specifically with the C-termnalnoncatalytic domain of NIMA in yeast. To determine if there was a stableinteraction between Pin1 and NIMA in HeLa cell extracts, recombinantPin1 was coupled to beads to determine whether NIMA could be recoveredfrom lysates of cells transiently expressing different NIMA mutants.Since NIMA induces mitotic arrest (Lu and Hunter, Cell, 81:413, 1995a),it is difficult to express sufficient wild type NIMA to detect complexformation, but since Pin1 interacted with the kinase-negative K40M NIMAas efficiently as wild type NIMA in the yeast two-hybrid system, we usedK40N NIMA and truncated derivative mutant constructs were used.

FIG. 5 shows immunoprecipitations to show the interaction between Pin1and the C-terminal noncatalytic domain of NIMAs in HeLa cells. FIG. 5Ashows the expression and purification of His-Pin1. The PIN1 cDNA wassubcloned into pProEx1 and the recombinant protein was purified frombacteria using Ni²⁻-NTA agarose column, followed by analyzing theprotein on a 15% SDS-containing gel, and Coomassie staining. Thepositions of His-Pin1 and standard size markers are indicated. FIG. 5Bshows the expression of NIMAs in HeLa cells. tTA-1 cells weretransfected with different FLAG-tagged NIMA constructs and labeled with³⁵S Express Label for 24 hr, followed by immunoprecipitation using theM2 mAb. The precipitated proteins were analyzed on a 10% SDS-containinggel followed by autoradiography. The positions of K40M, NIMA, K40MNIMA280, NIMA280-699 and standard size markers are indicated. FIG. 5Cshows a stable complex between recombinant Pin1 and NIMAs. Cell lysatessimilar to those in panel B were incubated with His-Pin1-Ni²⁻-NTA bead,as indicated in panel A and washed, followed by electrophoresis on anSDS-containing gel and autoradiography. FIGS. 5D and 5E showcoimmunoprecipitation of K40M NIMA with Pin1. tTA-1 cells werecotransfected with FLAG-tagged NIMA and HA-PIN1 constructs for 24 hr.Cell lysates were immunoprecipitated with the HA-specific 12CA5 mAb,followed by immunoblotting using the M2 mAB specific for the FLAG tag(D) and vice versa (E).

Out of several different fusion proteins tested, His-Pin1 was found tobe most stable and easiest to purify in large quantities when expressedin bacteria (FIG. 5A). When incubated with 35S-labeled extracts fromcells transiently expressing the full length K40M NIMA, an N-terminalfragment K40M NIMA280, or a C-terminal fragment NIMA280-699 (FIG. 5B),the recombinant His-Pin1 beads specifically bound K40M NIMA andNIMA280-699, but not K40M NIMA280 (FIG. 5C). Results also showed thatpurified GST-NIMA 280-699 directly interacts with purified Pin1 invitro. These results indicate that the recombinant Pin1 interacts withC-terminal domain of NIMA in the HeLa cell extracts.

To examine whether a Pin1 and NIMA complex is formed in the cell, an HAepitope tag was inserted at the N-terminus of Pin1 and coexpressed itwith FLAG-tagged NIMA constructs in HeLa cells using atetracycline-responsive expression system, as described previously (Luand Hunter, Cell, 81:413, 1995a). When cell lysates wereimmunoprecipitated with the HA tag-specific mAb (12CA5), followed byimmunoblot analysis using the M2mAb specific for the FLAG tag and viceversa, K40M NIMA but not K40M NIMA280 was detected in Pin1immunoprecipitates (FIG. 5D). Conversely, Pin1 was detected only in K40MNIMA immunoprecipitates (FIG. 5E). When the isolated catalytic domain ofNIMA was expressed in HeLa cells, it was located in the cytosol (Lu andHunter, Cell, 81:413, 1995a), whereas Pin1 is a nuclear protein (seebelow); therefore it is unlikely that Pin1 interacts with the NIMAcatalytic domain. These results, together with the in vitro bindingassay and the two-hybrid analysis, suggest that the C-terminalnoncatalytic domain of NIMA is involved in the interaction with Pin1.

To examine whether Pin1 affected NIMA kinase activity, purifiedrecombinant Pin1 protein was added to a NIMA kinase reaction mixture inthe presence of absence of the PL1 peptide substrate. Under theseconditions tested, Pin1 was neither phosphorylated by nor inhibited NIMAkinase. Similar results were obtained with several other protein kinasesincluding cyclin B/CDC2, ERK1 and PKA. These results indicated that Pin1is unlikely to act as a substrate or an inhibitor of the NIMA, CDC2,EPK1 and PKA protein kinases.

EXAMPLE 6 Pin1 Colocalizes With NIMA in a Defined Nuclear Substructure

To examine whether Pin1 is colocalized with Nima in human cells, thesubcellular localization of Pin1 was determined using indirectimmunofluorescence staining as described previously (Lu and Hunter,Cell, 81:413, 1995a). When Ha-tagged Pin1 was expressed in HeLa cellsand stained with the 12A5 mAB, Pin1 was observed almost exclusively inthe nucleus. This result indicates that Pin1 is a nuclear protein,consistent with the presence of a nuclear localization signal in Pin1(FIG. 2A). Confocal microscopy further revealed that although Pin1 wasdistributed throughout the nucleus, it was highly concentrated incertain areas with a speckled appearance (FIG. 6, right panel).

FIG. 6 shows the colocalization of Pin1 and NIMA and its kinase-negativemutant in HeLa Cells. Twenty four hr after transfection with the vectorsexpressing HA-tagged Pin1 only (right panels) or HA-tagged Pin1 andFLAG-tagged NIMA (left panels) or its kinase-negative mutant (K40M NIMA,middle panels), the transfeected tTA-1 cells were processed for indirectimmunofluorescence staining and examined by confocal microscopy. Toppanels: staining pattern for NIMA or K40M NIMA obtained with FLAGtag-specific M2 mAb or SC-35 obtaining with anti-SC-35 mAb, and thenFITC-conjugated IgG1-specific secondary antibodies; middle panels:staining pattern for Pin1 obtaining with HA tag-specific 10CA5 mAb andTexas-Red-conjugated IgG2b-specific secondary antibodies; bottom panels:double-staining for Pin1 and NIMA. Pin1 and K40M NIMA, or Pin1 and SC-35displayed by superimposing the respective top and middle images, withyellow color indicating colocalization. Arrows point to an untransfectedcell, indicating very little cross reactivity among the antibodies, Bar1 μm.

To examine whether Pin1 and NIMA were colocalized in HeLa cells,HA-tagged PIN1 and Flag-tagged nimA or its kinase-negative mutant (K40MnimA) cotransfected into tTA-1 cells and then examined the localizationof expressed proteins using tag-specific 12CA5 and M2 mAbs andisotope-specific secondary antibodies. In cells with the NIMA-inducedmitotic phenotype (FIG. 6, left panels), Pin1 was associated with thecondensed chromatin, but was also distributed throughout the cell, aswas NIMA (Lu and Hunter, 1995a). In K40M NIMA-expressing cells, Pin1 wasconcentrated in nuclear substructures, where the mutant NIMA was alsoconcentrated (FIG. 6, middle panels). The colocalization was extensive,but not complete (FIG. 6, middle lower panel). Pin1 was also colocalizedwith the C-terminal NIMA280-699 in the nuclear substructures. Sincesimilar nuclear substructures have been observed by immunostaining withanti-SC35 splicing factor (Fu and Maniatis, Nature, 343:437, 1990) andanti-PML antibodies (Dyck et al., Cell, 76:333, 1994), Pin1 nuclearsubstructures were examined to determine whether they were the same asthose localized by SC35 or PML. When cells expressing Pin1 were doublystained with 12CA5 and anti-splicing factor SC35 or anti-PML antibodies,the speckles displayed by Pin1 were found to be the same as thosedetected using anti-SC35 (FIG. 6, right panels), but not anti-PMLantibodies. These results indicate that Pin1 and K40M NIMA arecolocalized in the spliceosome nuclear substructure. Since usingidentical protocols, completely different localization patterns wereobserved for several other proteins, including the human NLK1 which wascolocalized with Pin3 in the nucleolus, the localization pattern of Pin1and NIMA is unlikely to be due to a nonspecific accumulation in certainareas of the nuclease resulting from over expression. Therefore, theseresults demonstrate that Pin1 is colocalized with NIMA in the nucleus ina defined nuclear substructure.

EXAMPLE 7 Pin1 Overexpression Inhibits NIMA-Induced Mitotic Arrest andInduces G2 Arrest in HeLa Cells

In the yeast two-hybrid system, expression of Pin1 rescued the lethalphenotype of NIMA, indicating that Pin1 might inhibit the mitoticfunction of NIMA. To examine whether over expression of Pin1 in HeLacells blocked NIMA-induced mitotic arrest, cells were contransfectedwith expression vectors for nimA and either PIN1 or an expression vectorcontrol and their phenotypes examined, as described previously (Lu andHunter, Cell, 81:413, 1995a).

FIG. 7 shows overexpression of PIN1 delays NIMA-induced mitotic arrestand induces a specific G2 arrest in HeLa cells. FIG. 7A shows theresults og tTA-1 cells cotransfected with nimA and PIN1 or controlvector. At the times indicated, cells were fixed and doubly labeled withM2 mAb and Hoechst dye, followed by scoring for the percentage of cellswith rounding and chromatin condensation in a sample of at least 250,NIMA-expressing cells. The data are the average from two independentexperiments. FIGS. 7B, C, and D show after transfection with PIN1expression vector or the control vector for 48 hr, tTA-1 cells werestained with the 12CA5 mAb and then with FITC-conjugated secondaryantibodies and propidium iodide, followed by FACS analysis. Based on theFITC intensity of the PIN1-transfected cells, cells were divided intotwo populations with 12CA5-negative (B) or -positive cells (C), and cellcycle profiles were determined to compare with those in totalvector-transfected cells (A).

At 24hr after transfection, ˜80% of NIMA-expressing cells were roundedup and contained highly condensed chromatin (FIG. 7A). When cells werecontransfected with nimA and PIN1, NIMA was expressed at much higherlevels than in cells contransfected with nimA and control vector, asdetected by immunofluorescence microscopy. However, at 24 hr only ˜21%of NIMA-expressing cells showed the complete mitotic phenotype. Theother NIMA-expressing cells were not completely rounded up and theirchromatin was not as condensed as those in cells expressing only NIMA(FIG. 5 left panels and 7A), although by 36 hr all the NIMA-expressingcells displayed the mitotic phenotype (FIG. 7A). These results indicatedthat Pin1 can partially inhibit the mitosis-promoting function of NIMA.

To examine whether overexpression of Pin1 blocked the G2/M transition.HeLa cells were transfected with either PIN1 or the control vector andcycle progression was examined by immunofluorescence microscopy and FACSanalysis. Cells expressing Pin1 contained a large interphase nucleus,and it was very difficult to find mitotic cells expressing Pin1,suggesting that Pin1 might induce a G2 arrest. FACS analysis indicatedthat the percentage of cells with 4 n DNA content was significantlyincreased in cells expressing Pin1 (12CA5-positive) (FIG. 7D). About 50%of cells were in G2, with commensurately fewer cells in G1 and S.Similar results were also obtained with over expression of anotherindependent PIN1 clone (H6) encoding a 5 amino acid N-terminal deletionmutant. In contrast, the vector-transtected cells showed very little12CA5 staining and displayed a similar cell cycle profile to that ofcells not expressing Pin1 (12CA5-negative) in the Pin1-transfected cellpopulation (FIGS. 7B and C). These results, together with the delayingeffect of Pin1 on NIMA-induced mitotic arrest, suggest that Pin1 caninhibit the NIMA pathway which is required for the G2/M transition.

EXAMPLE 8 PIN1 is a Functional Homologue of the Essential ESS1 Gene ofS. cerevisiae

Overexpression of Pin1 inhibits the G2/M transition in HeLa cells asshown above. If this inhibitory effect is due to the fact that highlevels of Pin1 affect a cell cycle checkpoint control, depletion of Pin1should promote the G2/M transition, resulting in a mitotic arrest. Toexamine this possibility, budding yeast were used where endogenousprotein expression can be readily manipulated. Since Pin1 shows strikingsimilarity to the yeast Ess1, the human PIN1 was tested to determinewhether it might functionally substitute for the yeast gene. Since ESS1knockout mutations are lethal in yeast (Hanes et al., Yeast, 5:55,1989), the ability of PIN1 to restore viability to ess1-mutants wasasicned. First, plasmids that express PIN1 under the control of aconstitutive yeast promoter were introduced into diploid cells in whichone copy of ESS1 is disrupted (ess1::URA3/ESS1). Cells were induced toundergo sporulation, tetrads were dissected, and viability of theresulting haploid spores was scored (Table 1A). As expected, tetradsderived from cells transforned with vector alone showed a 2:2segregation for spore viability (viable:inviable). In contrast, ˜25% ofthe tetrads from cells transformed with a PIN1-expressing plasmid showed4:0 seregation for spore viability, indicating that PIN1 complements theess1 mutant to allow spore outgrowth and haploid cell viability. PIN1did not complement ess1 mutant to allow spore outgrowth and haploid cellviability. PIN1 did not complement ess1 mutants quite as efficiently asESS1 itself, where more than half the tetrads from cells bearing an ESS1plasmid showed a 4:0 segregation from spore viability. Growth curvesrevealed that PIN1-expressing ess1 cells had doubling times onlyslightly longer than control cells. Thus, human PIN1 can functionallysubstitute for ESS1 in haploid yeast cells.

Second, the PIN1 expression plasmid was introduced into diploid cells inwhich both chromosomal copies of ESS1 are disrupted(ess1::URA3/ess1::URA3) but that remain viable by maintaining aplasmid-borne copy of ESS1. If PIN1 functionally substitute for ESS1,then it should be possible to cure cells of the ESS1 plasmid. Table 1Bshows that cells serially passaged in media selecting only for the PIN1plasmid (LEU2) lost the ESS1 plasmid (HIS3) about 12% of the time,whereas cells containing a control vector did not lose the ESS1 plasmid.Thus, human PIN1 can functionally substitute for ESS1 in diploid yeastcells. TABLE 1 PIN1 Complements the ESS1 Knockout Mutation in BuddingYeast A. Tetrad Analysis Total Number of Viable Spores per TetradPlasmid Tetrads 0 1 2 3 4 Vector 21 0 3 17 1 0 PIN1 83 5 6 46 6 20 ESS129 0 0 6 6 17 B. Curing Experiment Plasmid His⁺/Leu⁺ Ura⁺ Loss of PIN1plasmid (%) Vector 216/216 216 0 PIN1 380/431 431 12A. PIN1 rescues ess1 lethality in haploids. The heterozygous disruptionstrain MGGS3/pSH-U was transformed with an ESS1- or PIN1-expressionvector plasmid or control vector. Cells were induced to undergosporulation and tetrads were dissected. The number of viable sporesincluded only those that showed proper segregation of independentmarkers. As expected, almost all segregants that were ura⁺ (i.e. carriedthe ess1::URA3 knockout allele) were also his⁺ or leu⁺ indicating thepresence of the ESS or PIN1 containing plasmid, respectively.B. PIN1 permits loss of ESS1-containing plasmids in a ess1 diploidknockout strain. A diploid disruption strain (ess1::URA3/ess1::URA3)carrying the ESS1 -plasmid (HIS3) was transformed with the vector alone(LEU2) or a PIN1-expression vector plasmid (LEU2). Cells were seriallypassaged in leucine-deficient media that maintains selection for controlor the PIN1 plasmid but not for the ESS1 plasmid. Cells were plated andphenotypes of individual colonies scored by replica plating toappropriate selective media. Loss of the ESS1 plasmid is detected byloss of the his⁺ phenotype, while the presence of PIN1 is detected by aleu⁺ phenotype. A ura⁺ phenotype confirms the presence of the knockoutallele (ess1::URA3).

EXAMPLE 9 Depletion of PIN1/ESS1 Results in Mitotic Areest and NuclearFragmentation in S. Cerevisiae

To examine the effect of depleting Pin1/Ess1 on the cell cycle, PIN1driven by the regulated GAL promoter was introduced into an ess1 yeaststrain. As expected, Pin1-expressing strains grew normally in inducingmedia (galactose) or noninducing media (galactose/glucose, with a basallevel of PIN1 expression), but did not grow in repressing media(glucose).

FIG. 8 shows depletion of Pin1/Ess1 results in mitotic arrest andnuclear fragmentation in yeast. A Pin1-dependent strain (YSH12.4) wasshifted from inducing media to repressing media, harvested and fixedwith 70% ethanol at the times indicated. The cells were stained withDAPI or propidium iodide, followed by videomicroscopy under Nomarksi(DIC) or fluorescent (DAPI) illumination, or FACS analysis,respectively. The bar is 10 μm and the inserts show a highermagnification of a representative cell.

Cells depleted of Pin1 following the shift to repressing media displayeda striking terminal phenotype indicating mitotic arrest (Table 2, FIG.8). Following about 6 hr of normal growth, cell division was inhibitedand by 12 hr, cells began to accumulate in mitosis as judged by theincreasing percentage of cells containing a large bud (dumbbell shape)and by FACS analysis (Table 2, FIG. 8, middle panels). As revealed byDAPI staining, a high percentage of cells containing nuclear stainingmaterial in the neck between the mother and the bud, suggesting thatmitotic chromosome segregation was slowed or inhibited. Control strainsthat carried GAL::PIN1, but were wild-type for ESS1, showed normaldistribution of cells throughout the cell cycle as judgedmicroscopically and by FACS analysis. By 24 hr., cell division stoppedand most cells were arrested in mitosis. Interestingly, cells depletedof Pin1 for extended periods of time (18-30 hr) showed multiple nuclearfragments, which appeared randomly distributed throughout the cell (FIG.8, right panels). FACS analysis revealed that cells with a 2 n DNAcontent accumulated over time, with most cells containing 2 n DNAcontent by 24 hr after the shift to repressing media (FIG. 8, bottompanels). When cells were shifted from noninducing media for expressingmedia, this phenotypes appeared earlier. Cells were arrested at mitosisby 12 hr with a mitotic chromatin located in the neck in about 40% ofcells and nuclear fragmentation in most other cells, indicating thedependence of the phenotypes on the initial level of Pin1 expression.These results show that depletion of Pin1 results in mitotic arrest andeventually nuclear fragmentation; phenotypes similar to those observedin Aspergillus and HeLa cells that overproduce NIMA (Osmain et al.,Cell, 53:237, 1988; O'Connell et al., EMBO J., 13:4926, 1994; Lu andHunter, Cell, 81, 413, 1995a). TABLE 2 Depletion of Pin1/ESS1 Results inMitotic Arrest in Budding Yeast Time after % Unbudded % Small Bud %Medium Bud % Large Bud Shift (hr) (G1) (S) (G2) (M) 0 39.2 21.2 18.621.0 6 27.2 22.2 24.8 26.0 12 32.2 10.2 14.4 43.2 18 26.0 6.8 9.0 58.224 20.2 3.6 7.4 68.8 30 17.6 3.8 4.8 73.8A Pin1-dependent strain (YSH 12.4) was shifted from inducing media torepressing media, harvested and fixed with ethanol at the timesindicated. Bud size distribution was scored under Nomarski illuminationafter cells were stained with DAP1 and sonicated. About 500 cells werecounted for each point.

SUMMARY

Using the yeast two-hybrid system, the present invention identifies thefirst NIMA-interacting protein of human origin Pin1. Pin1 contains anN-terminal WW domain and a C-terminal PPIase domain, and has PPIaseactivity in vitro. PIN1 functionally rescues a knockout mutation of theESS1 gene, which is essential for the growth of budding yeast cells.These results indicate that Pin1 is conserved from yeast to human;Pin1/Ess1 is the first PPIase knows to be essential for life. Pin1interacts with the C-terminal noncatalytic domain of NIMA andcolocalizes with NIMA in a defined nuclear substructure in HeLa cells.Whereas overexpression of Pin1 induces a specific G2 arrest, and delaysNIMA-induced mitosis in HeLa cells, depletion of Pin1/Ess1 triggersmitotic arrest and nuclear fragmentation in budding yeast. These resultsindicate that Pin1 regulates entry into mitosis, probably via the NIMAmitotic pathway.

The primary structure of Pin1 contains two identifiable domains. TheC-terminal two-thirds of Pin1 contains residues that are highlyconserved in the newly delineated family of PPIases that includesparvulin, PrsA, SurA, NifM, PrtM, Cbf2 and Ess1 (Rudd, et al., TIBS,20:12, 1995). PrsA, SurA, NifM, PrtM have been shown to be involved inmaturation and/or transport of specific proteins or protein classes.Parvulin, orininally identified during a chromatographic purificationprocedure, is the prototype of this family, it contains 96 amino acidsand catalyzes the cis/trans isomerization of X-Pro peptide bonds, evenin the presence of immunosuppressive drugs (Rahfeld et al., FEBS Lett.,352:180, 1994a and FEBS Lett., 343:65, 1994b). The homology between Pin1and parvulin spans almost the entire parvulin molecule, stronglysugaesting that Pin1 is a PPIase. The in vitro PPIase assay shown in theabove Examples confirms that Pin1 can indeed catalyze the cis/transisomerization of peptidyl-prolyl peptide bonds.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without deporting from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1-3. (canceled).
 4. An isolated nucleic acid comprising at least about15 contiguous nucleotides of a nucleotide sequence substantially thesame as nucleotides 13-129 of SEQ ID NO: 1, or compliment thereof. 5.The isolated nucleic acid of claim 4, wherein said nucleotide sequenceselectively hybridizes to a fragment of nucleotides 13-129 of SEQ IDNO:1.
 6. The isolated nucleic acid of claim 4, comprising a nucleotidesequence encoding a portion of a Pin1 polypeptide having substantiallythe same amino acid sequence as amino acids 5-43 of SEQ ID NO:2.
 7. Theisolated nucleic acid of claim 6, comprising a nucleotide sequenceencoding a portion of a Pin1 polypeptide having substantially the sameprotein-protein interaction activity as amino acids 5-43 of SEQ ID NO:2.8. The isolated nucleic acid of claim 4, comprising a degeneratenucleotide sequence variant encoding a portion of a Pin1 polypeptidehaving substantially the same amino acid sequence as amino acids 5-43 ofSEQ ID NO:2.
 9. The isolated nucleic acid of claim 4, comprising thesame nucleotide sequence as nucleotides 13 -129 of SEQ ID NO:1.
 10. Anisolated nucleic acid comprising a nucleotide sequence substantially thesame as nucleotides 13 -129 of SEQ ID NO:1.
 11. The isolated nucleicacid of claim 10, wherein said nucleotide sequence selectivelyhybridizes to nucleotides 13-129 of SEQ ID NO:1.
 12. The isolatednucleic acid of claim 10, comprising a nucleotide sequence encoding aPin1 polypeptide having substantially the same amino acid sequence asamino acids 5-43 of SEQ ID NO:2.
 13. The isolated nucleic acid of claim12, comprising a nucleotide sequence encoding a Pin1 polypeptide havingsubstantially the same protein-protein interaction activity as aminoacids 5-43 of SEQ ID NO:2.
 14. The isolated nucleic acid of claim 10,comprising a degenerate nucleotide sequence variant encoding a Pin1polypeptide having substantially the same amino acid sequence as aminoacids 5-43 of SEQ ID NO:2.
 15. The isolated nucleic acid of claim 10,comprising the same nucleotide sequence as nucleotides 13 -129 of SEQ IDNO:
 1. 16. An isolated nucleic acid comprising a nucleotide sequenceencoding a portion of SEQ ID NO:2 that exhibits protein-proteinassociation activity.
 17. The isolated nucleic acid of claim 16, whereinsaid protein-protein association activity comprises NIMA mitotic kinasebinding activity.
 18. An isolated nucleic acid comprising at least about15 contiguous nucleotides of a nucleotide sequence substantially thesame as nucleotides 175-489 of SEQ ID NO:1.
 19. The isolated nucleicacid of claim 18, wherein said nucleotide sequence selectivelyhybridizes to a fragment of nucleotides 175-489 of SEQ ID NO:1.
 20. Theisolated nucleic acid of claim 18, comprising a nucleotide sequenceencoding a portion of a Pin1 polypeptide having substantially the sameamino acid sequence as amino acids 59-163 of SEQ ID NO:2.
 21. Theisolated nucleic acid of claim 20, comprising a nucleotide sequenceencoding a portion of a Pin1 polypeptide having substantially the samePPIase activity as amino acids 59-163 of SEQ ID NO:2.
 22. The isolatednucleic acid of claim 18, comprising a degenerate nucleotide sequencevariant encoding a portion of a Pin1 polypeptide having substantiallythe same amino acid sequence as amino acids 59-163 of SEQ ID NO:2. 23.The isolated nucleic acid of claim 18, comprising the same nucleotidesequence as nucleotides 175-489 of SEQ ID NO:1.
 24. An isolated nucleicacid comprising a nucleotide sequence substantially the same asnucleotides 175-489 of SEQ ID NO:1.
 25. The isolated nucleic acid ofclaim 24, wherein said nucleotide sequence selectively hybridizes tonucleotides 175-489 of SEQ ID NO:1.
 26. The isolated nucleic acid ofclaim 24, comprising a nucleotide sequence encoding a Pin1 polypeptidehaving substantially the same amino acid sequence as amino acids 59-163of SEQ ID NO:2.
 27. The isolated nucleic acid of claim 26, comprising anucleotide sequence encoding a Pin1 polypeptide having substantially thesame PPIase activity as amino acids 59-163 of SEQ ID NO:2.
 28. Theisolated nucleic acid of claim 24, comprising a degenerate nucleotidesequence variant encoding a Pin1 polypeptide having substantially thesame amino acid sequence as amino acids 59-163 of SEQ ID NO:2.
 29. Theisolated nucleic acid of claim 24, comprising the same nucleotidesequence as nucleotides 175-489 of SEQ ID NO:1.
 30. An isolated nucleicacid comprising a nucleotide sequence encoding a portion of SEQ ID NO:2that exhibits PPIase activity.